Continuous Directed Evolution System for Protein Diversification
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Solution Overview
Problem
Current methods for diversifying nucleic acids and proteins are limited by the number of recombination events and library size, with existing continuous evolution systems being non-generalizable and slow, lacking an intrinsic means for functional mutant selection and transmission between cells.
Innovation Solution
A continuous directed evolution system that enables nucleic acid transfer between cells in a function-dependent manner using phage or viral infection, with engineered host cells capable of screening for specific functions and utilizing helper and accessory plasmids to facilitate phage replication and selection, allowing for continuous diversification and selection of functional proteins and RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If nonhomologous recombination methods are used to diversify proteins, then recombination events can occur at defined sites without sequence homology, but the number of recombination events is limited (three or fewer per 500 bp)
Solution Approach 1:
The patent implements continuous evolution systems where mutagenesis and selection occur continuously rather than in discrete steps. The system maintains a continuous population of evolving molecules through constant replication and selection pressure, enabling sustained generation of diversity without the interruptions inherent in traditional batch methods.
Solution Approach 2:
The evolved molecules themselves perform the selection function through their functional activity. The system uses functional complementation where evolved proteins or nucleic acids directly contribute to the survival or propagation of their encoding sequences, eliminating the need for external selection interventions and enabling autonomous continuous evolution.
2Measurement precision
If traditional directed evolution methods are used, then functional mutants can be selected through discrete rounds of selection, but the process is slow and library size is limited
Solution Approach 1:
The patent implements continuous evolution systems where mutagenesis and selection occur continuously rather than in discrete steps. The system maintains a continuous population of evolving molecules through constant replication and selection pressure, enabling sustained generation of diversity without the interruptions inherent in traditional batch methods.
Solution Approach 2:
The patent uses functional complementation as an intermediary mechanism where evolved molecules contribute to the survival or propagation of their encoding sequences. This intermediary system enables continuous selection pressure to be applied to large libraries without requiring manual intervention at each selection step, thereby scaling the process while maintaining selection accuracy.
3Quantity of substance
If error-prone PCR is used to generate diversity, then nucleic acid variants can be produced, but the number of functional mutants with novel properties is limited
Solution Approach 1:
The patent implements continuous evolution systems where mutagenesis and selection occur continuously rather than in discrete steps. The system maintains a continuous population of evolving molecules through constant replication and selection pressure, enabling sustained generation of diversity without the interruptions inherent in traditional batch methods.
Solution Approach 2:
The patent employs continuous selection pressure as feedback to guide the evolution process. The functional activity of evolved molecules directly influences their propagation, creating a feedback loop where only functional variants are enriched. This continuous feedback mechanism ensures that diversity generation is coupled with functional validation, increasing the proportion of functional mutants with novel properties.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This system enables rapid and efficient evolution of nucleic acids and proteins, overcoming limitations on library size and number of rounds of selection, allowing for the exploration of a vast sequence space and achieving significant increases in evolutionary efficiency, with the potential to evolve diverse functionalities in proteins and RNA.
Implementation Method 1
introducing at least one of the mutated functional nucleic acid strands into a second host cell, wherein the introducing comprises phage or viral infection
Data Source
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AI summary
The present invention discloses generalizable methods of evolving nucleic acids and proteins utilizing continuous directed evolution. The invention discloses methods of passing a nucleic acid from cell to cell in a desired function-dependent manner. The linkage of the desired function and passage of the nucleic acid from cell to cell allows for continuous selection and mutation of the nucleic acid.